The present invention relates, in general, to methods of inducing a therapeutic effect. In particular, the present invention relates to therapeutic uses of Raf protein kinases.
Raf serine- and threonine-specific protein kinases are cytosolic enzymes that stimulate cell growth in a variety of cell systems (Rapp, U. R., et al. (1988) In The oncogene handbook; T. Curran, E. P. Reddy, and A. Skalka (ed.) Elsevier Science Publishers; The Netherlands, pp.213-253; Rapp, U. R., et al. (1988) Cold Spring Harbor Sym. Quant. Biol. 53:173-184; Rapp, U.R., et al. (1990) In: Curr. Top. Microbiol. Immunol. Potter and Melchers (eds), Berlin, Springer-Verlag 166:129-139). Three isozymes have been characterized: c-Raf (Raf-1) (Bonner, T. I., et al. (1986) Nucleic Acids Res. 14:1009-1015 see FIG. 9). A-Raf (Beck, T. W., et al. (1987) Nucleic Acids Res. 15:595-609 see FIG. 10), and B-Raf (Ikawa, S., et al. (1988) Mol. Cell. Biol. 8:2651-2654; Sithanandam, G. et al. (1990) Oncogene 5:1775 see FIG. 11). These enzymes differ in their expression in various tissues. Raf-1 is expressed in all organs and in all cell lines that have been examined, and A- and B-Raf are expressed in urogenital and brain tissues, respectively (Storm, S. M. (1990) Oncogene 5:345-351).
Raf genes are proto-oncogenes: they can initiate malignant transformation of cells when expressed in specifically altered forms. Genetic changes that lead to oncogenic activation generate a constitutively active protein kinase by removal or interference with an N-terminal negative regulatory domain of the protein (Heidecker, G., et al. (1990) Mol. Cell. Biol. 10:2503-2512; Rapp, U. R., et al. (1987) In Oncogenes and cancer S. A. Aaronson, J. Bishop, T. Sugimura, M. Terada, K. Toyoshima, and P. K. Vogt (ed.) Japan Scientific Press, Tokyo). Microinjection into NIH 3T3 cells of oncogenically activated but not wild-type versions of the Raf-protein prepared with Escherichia coli expression vectors results in morphological transformation and stimulates DNA synthesis (Rapp, U. R., et al. (1987) In Oncogenes and cancer; S. A. Aaronson, J. Bishop, T. Sugimura, M. Terada, K. Toyoshima, and P. K. Vogt (ed.). Japan Scientific Press, Tokyo; Smith, M. R., et al. (1990) Mol. Cell. Biol. 10:3828-3833). Thus, activated Raf-1 is an intracellular activator of cell growth. Raf-1 protein serine kinase is a candidate downstream effector of mitogen signal transduction, since Raf oncogenes overcome growth arrest resulting from a block of cellular ras activity due either to a cellular mutation (ras revertant cells) or microinjection of anti-ras antibodies (Rapp, U. R., et al. (1988) In The Oncogene Handbook, T. Curran, E. P. Reddy, and A. Skalka (ed.), Elsevier Science Publishers; The Netherlands, pp.213-253; Smith, M. R., et al. (1986) Nature (London) 320:540-543).
c-Ras function is required for transformation by a variety of membrane-bound oncogenes and for growth stimulation by mitogens contained in serum (Smith, M. R., et al. (1986) Nature (London) 320:540-543). Raf-1 protein merine kinase activity is regulated by mitogens via phosphorylation (Morrison, D. K., et al. (1989) Cell 58:648-657), which also effects subcellular distribution (Olah, Z., et al. (1991) Exp. Brain Res.84:403; Rapp, U. R., et al. (1988) Cold Spring Harbor Sym. Quant. Biol. 53:173-184).
Raf-1 activating growth factors include platelet-derived growth factor (PDGF) (Morrison, D. K., et al. (1988) Proc. Natl. Acad. Sci. USA 85:8855-8859) colony-stimulating factor 1 (Baccarini, M., et al. (1990) EMBO J. 9:3649-3657), insulin (Blackshear, P. J., et al. (1990) J. Biol. Chem. 265:12131-12134; Kovacina, K. S., et al. (1990) J. Biol. Chem. 265:12115-12118), epidermal growth factor (EGF) (Morrison, D. K., et al. (1988) Proc. Natl. Acad. Sci. USA 85:8855-8859), interleukin 2 (Turner, B. C. et al. (1991) Proc. Natl. Acad. Sci. USA 88:1227), and interleukin 3 and granulocyte-macrophage colony-stimulating factor (Carroll, M. P., et al (1990) J. Biol. Chem. 265:19812-19817). Upon mitogen treatment of cells, the transiently activated Raf-1 protein serine kinase translocates to the perinuclear area and the nucleus (Olah, Z., et al. (1991) Exp. Brain Res. 84:403; Rapp, U. R., et al. (1988) Cold Spring Harbor Sym. Quant. Biol. 53:173-184). Cells containing activated Raf are altered in their pattern of gene expression (Heidecker, G., et al. (1989) In Genes and signal transduction in multistage carcinogenesis, N. Colburn (ed.), Marcel Dekker, Inc., New York. pp. 339-374), and Raf oncogenes activate transcription from Ap-1/PEA3-dependent promoters in transient transfection assays (Jamal, S., et al. (1990) Science 344:463-466; Kaibuchi, K., et al. (1989) J. Biol. Chem. 264:20855-20858; Wasylyk, C., at al. (1989) Mol. Cell. Biol. 9:2247-2250).
There are at least two independent pathways for Raf-1 activation by extracellular mitogens: one involving protein kinase C (PKC) and a second initiated by protein tyrosine kinases (Blackshear, P. J., et al. (1990) J. Biol. Chem. 265:12131-12134; Kovacina, K. S., et al. (1990) J. Biol. Chem. 265:12115-12118; Morrison, D. K., et al. (1988) Proc. Natl. Acad. Sci. USA 85:8855-8859; Siegel, J. N., et al.(1990) J. Biol. Chem. 265:18472-18480; Turner, B. C. et al. (1991) Proc. Natl. Acad. Sci. USA 88:1227). In either case, activation involves Raf-1 protein phosphorylation. Raf-1 phosphorylation may be a consequence of a kinase cascade amplified by autophosphorylation or may be caused entirely by autophosphorylation initiated by binding of a putative activating ligand to the Raf-1 regulatory domain, analogous to PKC activation by diacylglycerol (Nishizuka, Y. (1986) Science 233:305-312).
It is a general object of this invention to provide a construct comprising a DNA segment comprising a Raf gene in an antisense orientation downstream of a promoter.
It is a specific object of this invention to provide a method of inhibiting Raf expression comprising expressing an antisense Raf gene in a cell such that said Raf expression is inhibited.
It is a further object of the invention to provide a method of inhibiting Raf kinase activity comprising replacing a serine or threonine amino acid within the Raf gene for an amino acid not susceptible to phosphorylation.
Further objects and advantages of the present invention will be clear from the description that follows.